This invention relates to suspension cultures of mammalian cells and, more particularly, to the agitated liquid suspension culturing of simian virus 40 transformed 3T3 mouse embryo cells (SV3T3) to produce the physiologically active substances MIF and TAF.
The in vitro viral transformation of cell lines by infecting an established cell line with virus is well known. Thus, the simian vacuolating virus (SV-40) is known to induce the transformation of various cell lines as reported by Black and Rowe, Proc. Soc. Exptl. Biol. & Med. 114, 721-27 (1963). The development of the established cell line 3T3 was first disclosed by Todaro and Green, J. Cell. Biol. 17, 299-313 (1963) and the transformation of this cell line by SV-40 to alter permanently its properties was further reported by Todaro et al, Proc. Nat. Acad. Sci. 51, 66-73 (1964).
The culturing of SV3T3 cells is important for obtaining certain physiologically active products of the cell metabolism. Thus, it is known that these transformed fibroblasts elaborate a macromolecular product that inhibits macrophage migration known as migration inhibition factor (MIF). The nature of MIF produced by sensitized lymphocytes has been fully described by David, Proc. Natl. Acad. Sci. U.S. 56, 72-77 (1966) and Fed. Proceedings, 30, 1730-35 (1971). The production of MIF from SV3T3 cells in particular is disclosed by Hammond et al, Science 185, 955-57 (1974), and by Poste, Exptl. Cell Res. 92, 233-90 (1975).
Another substance obtained by the culturing of SV3T3 cells is the tumor angiogenesis factor (TAF) which is known to stimulate the proliferation of new capillaries. The biological characteristics and assay of TAF have been summarized by Folkman, Cancer Res. 34, 2109-2113 (1974) and by Folkman and Klagsbrun in Chapter 31 of "Fundamental Aspects of Neoplasia," at pages 401-412, edited by Gottlieb et al, Springer-Verlag, New York, 1975.
The production of both of the aforesaid substances, MIF and TAF, is desired for their respective uses in various investigations of tumor biology and for diagnostic and other medical purposes. Although MIF and TAF can be isolated from tissue grown in vivo, the in vitro production would provide a more adequate and uniform supply of these materials.
The SV3T3 cell is generally known as an adherent anchorage dependent cell; Gail and Boone, Exptl. Cell. Res. 70, 33-40 (1972). An anchorage dependent cell when cultured in vitro normally must anchor itself to a surface such as a glass or plastic surface before it can divide. As such, these cells ordinarily are grown as monolayers in vessels such as flasks and petri dishes. While such growth in monolayers often is desirable, an agitated liquid suspension culture of SV3T3 cells would afford significant advantages in growth and harvesting provided that the desired MIF and TAF products would continue to be elaborated by these cultures.
Growth of SV3T3 cells in suspension cultures of 1.2% Methocel (8000 centipoises) has been reported heretofore; Otsuka and Moskowitz, J. Cell Physiol. 85, 665-74 and 87, 213-40 (1975). However, these are viscous, unagitated (static) suspensions which do not provide the advantages of cell growth obtained by the agitated liquid suspension culture as defined herein. The nature and characteristics of such static suspension cultures are further described by Stoker et al, J. Cancer 3, 683-93 (1968).